猪流行性腹泻病毒TaqMan检测方法的建立及基于S基因的遗传变异分析

旨在建立一种能快速、高效且灵敏的检测出猪流行性腹泻病毒（PEDV）的通用型TaqMan qPCR检测方法，并进一步了解河北省PEDV的遗传变异情况。本研究参考PEDV变异株AJ1102的M基因的保守区域设计特异性引物及探针，对引物浓度、退火温度等条件进行优化，建立了一种应用于猪场筛查PEDV的qPCR方法，并对筛查出的阳性样品S基因扩增测序后进行遗传进化分析。结果显示：该方法的特异性强，与猪圆环病毒2型（PCV2）、猪瘟病毒（CSFV）及传染型胃肠炎病毒（TGEV）等猪病常见病原均无交叉反应；建立的检测方法对pMD19T-PEDV标准品的最低检测下限为1.09×10¹拷贝·μL^-1，比普通PCR灵敏约100倍，敏感性高；组内和组间的变异系数均小于1%，重复性良好。基于S全基因成功克隆出的9条序列分布于GⅡ的两个亚群，克隆的9条序列的核苷酸相似性为96.6%～99.1%；氨基酸的相似性为94.6%～98.7%。结果表明：本研究得到的PEDV流行株与近年来国内分离株的关系较为密切，而与中国目前使用的疫苗株以及欧洲株亲缘关系较远，这表明河北部分地区PEDV当前流行毒株比较复杂且有变异的趋势，提示持续检测PEDV流行毒株变异动态的必要性。本研究不仅为PEDV的临床诊断提供了技术支持，更为进一步掌握PEDV遗传进化规律提供了参考依据。

Establishment of TaqMan Detection Method of Porcine Epidemic Diarrhea Virus and Analysis of Genetic Variation based on S Gene

The present study established a universal TaqMan qPCR detection method that can quickly, efficiently and sensitively detect porcine epidemic diarrhea virus (PEDV), and further understand the genetic variation of PEDV in Hebei Province. Here we designed specific primers and probes with reference to the conservative region of M gene of the PEDV variant AJ1102, and optimized the primer concentration, annealing temperature and other conditions, a qPCR method for screening PEDV in pig farms was established, and the S gene of the positive samples was amplified and sequenced for genetic evolution analysis. The results showed that the specificity of this method was strong, and there was no cross reaction with common pig diseases such as porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3) and transmissible gastroenteritis virus (TGEV); The minimum detection limit of the established detection method for pMD19T-PEDV standard is 1.09×10¹ copy·μL^-1. It is about 100 times more sensitive than ordinary PCR and has high sensitivity; The coefficient of variation within and between groups was less than 1%, with good repeatability. The nine sequences successfully cloned based on the whole S gene were distributed in two subgroups of GⅡ, and the nucleotide homology of the nine cloned sequences were 96.6%-99.1%; The homology of amino acids were 94.6%-98.7%. The results showed that the epidemic strains of PEDV obtained in this study were closely related to the domestic strains isolated in recent years, but far related to the vaccine strains currently used in China and the European strains, which indicated that the current epidemic strains of PEDV in some areas of Hebei Province were more complex and had a trend of variation, suggesting the necessity of continuous detection of the variation dynamics of the epidemic strains of PEDV. This study not only provides technical support for the clinical diagnosis of PEDV, but also provides a reference for further understanding the genetic evolution of PEDV.